Element having bioactive substance fixed thereto

ABSTRACT

By using a biologically active substance-immobilized device, which comprises a base particle comprising a core particle and an organic compound having two or more hydrophilic groups and immobilized on the core particle by a chemical bond and a biologically active substance bonded to the base particle via the organic compound, a substance specifically bonding to the biologically active substance is detected or measured.

TECHNICAL FIELD

The present invention relates to a device for detection or measurementof a biologically active substance or for therapeutic treatment. Thedevice of the present invention relates to the fields of diagnosis,therapeutic treatment, biochemistry and so forth.

BACKGROUND ART

In analyses of nucleic acids based on hybridization, immunoassays and soforth, techniques of immobilizing nucleic acids or proteins on a carriersuch as particles, membranes and plates have conventionally beenutilized. As such methods for immobilizing biomolecules, the followingmethods are known for nucleic acids:

(1) A method of chemically bonding a nucleic acid introduced with amodification group, such as immobilization by a disulfide bond between anucleic acid having a thiol group at the 5′ end and a bead-like basematerial having thiol groups (P. J. R. Day et al., Biochem. J., vol.278, pp. 735-740 (1991));

(2) A method of immobilizing a nucleic acid by adsorption on a carriersuch as nitrocellulose, nylon membrane, or glass coated with a cationicpolymer such as poly-L-Lysine through ultraviolet (UV) irradiation orheat treatment (International Patent Publication in Japanese (Kohyo) No.10-503841; J. Sambrook et al., Molecular Cloning, Cold Spring HarborLaboratory Press, Second Edition, pp. 2.109-2.113 and pp. 9.34-9.46);

(3) A method of physically adsorbing a nucleic acid on wells of amicroplate treated with a polylysine solution by injecting the nucleicacid into the wells and heating the plate at 37° C. (G. C. N. Parry etal., Biochem. Soc. Trans., vol. 17, pp. 230-231 (1989));

(4) A method of synthesizing DNA on a base material by using nucleotidesbonded to the base material (WO97/10365);

(5) A method of chemically bonding a nucleic acid introduced with amodification group such as immobilization of a nucleic acid having abiotin group at the 5′ end on a magnetic bead carrier covered with astreptavidin-coated film (International Patent Publication in JapaneseNo. 2000-507806); and

(6) A method of immobilizing a nucleic acid on polystyrene beads coatedwith polycarbodiimide (carbodiimide groups) (Japanese Patent Laid-open(Kokai) No. 8-23975).

However, these methods suffer from drawbacks. That is, the method of (1)requires an extremely special apparatus and regents. Further, in themethods of (2) and (3), nucleic acids are dropped off from the carriersduring the hybridization, in particular, in operation processes, and asa result, detection sensitivity may be reduced, or reproducibilitycannot be obtained. Furthermore, with these methods, although a longnucleic acid can be immobilized, a short nucleic acid of about 50-mer orshorter such as oligomers cannot be efficiently immobilized. Further,the method of (4) also requires an extremely special apparatus andregents for synthesizing DNA on the base material, and the nucleic acidthat can be synthesized is limited to about 25-mer or shorter. Moreover,the method of (5) has drawbacks that the material of the base materialis limited, and storage stability of the nucleic acid-immobilized beadsis poor. In the method of (6), a nucleic acid is reacted with acarbodiimide group, and therefore the nucleic acid is not separated fromthe polycarbodiimide during hybridization. However, because thepolystyrene and the polycarbodiimide do not bond to each other withchemical bonds, the polycarbodiimide tends to separate from thepolystyrene beads during hybridization.

On the other hand, there have been conducted researches in which it isattempted to activate a monocarbodiimide and immobilize a substance on aparticle serving as a base material via an amide bond from an amino acidor the like and thereby improve dispersibility of the particles, asdescribed in Japanese Patent No. 2629909. However, because unnecessaryproducts such as urea derivatives are produced by the condensationreaction, a problem of time-consuming washing step arises. Otherproblems also arise, for example, the substance to be immobilized islimited to particular active substances capable of forming an amidebond, and the product may not exhibit performance as a test device ordiagnostic device depending on the bonding site (position) of particleserving as a base material and amino group or carboxyl group in an aminoacid.

DISCLOSURE OF THE INVENTION

An object of the present invention is to provide a device for detectionor measurement of a biologically active substance or for therapeutictreatment, which exhibits favorable stability in a dispersion medium,and a production method thereof.

The inventors of the present invention conducted various researches inorder to achieve the aforementioned object. As a result, they foundthat, by immobilizing a biologically active substance on particles usingan organic compound having two or more hydrophilic groups, dispersionstability of the particles in a solution could be improved, and thusaccomplished the present invention.

That is, the present invention provides the followings.

(1) A biologically active substance-immobilized device, which comprisesa base particle comprising a core particle and an organic compoundhaving two or more hydrophilic groups and immobilized on the coreparticle by a chemical bond and a biologically active substance bondedto the base particle via the organic compound.

(2) The device according to (1), which is used in an aqueous medium.

(3) The device according to (1) or (2), wherein the base particle has anaverage particle diameter of 0.01 to 100 μm.

(4) The device according to any one of (1) to (3), wherein the baseparticle has a spherical or substantially spherical shape.

(5) The device according to any one of (1) to (4), wherein at least oneof CV_(b) ratio and CV_(c) ratio defined by the following equations is0.6 to 3.0:CV _(b) ratio=CV ₁ /CV ₃CV _(c)ratio=CV ₂ /CV ₃CV ₁=(Standard deviation of core particle diameter/Average core particlediameter)×100CV ₂=(Standard deviation of base particle diameter/Average base particlediameter)×100CV ₃=(Standard deviation of device diameter/average device particlediameter)×100(6) The device according to any one of (1) to (5), wherein the coreparticle and the biologically active substance are bonded by a reactionwith a functional group selected from carbodiimide group, ester group,carbonate group, epoxy group and oxazoline group.(7) The device according to any one of (1) to (6), wherein the organiccompound is a compound represented by the following formula:A_(x)—(R—X)_(n)—R—A_(y)  (I)wherein A_(x) and A_(y) independently represent a segment having afunctional group that exhibits hydrophilicity and may be identical ordifferent, R independently represents an organic group of two or morevalences, X independently represents carbodiimide group, epoxy group oroxazoline group, and n is an integer of 2 to 80, preferably 2 to 40.(8) The device according to any one of (1) to (7), wherein thebiologically active substance is selected from a nucleic acid, protein,hapten and saccharide.(9) The device according to any one of (1) to (8), which is fordetecting or measuring a second biologically active substance containedin a sample by using a specific bond of the biologically activesubstance and the second biologically active substance in the sample.(10) The device according to any one of (1) to (8), wherein thebiologically active substance is an agent for therapeutic treatment of adisease.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereafter, the present invention will be explained in detail.

<1> Device of the Present Invention

The device of the present invention comprises a base particle comprisinga core particle and an organic compound having two or more hydrophilicgroups (hereinafter referred to as “organic compound A”) immobilized onthe core particle by a chemical bond and a biologically active substancebonded to the base particle via the organic compound A.

Hereafter, the device of the present invention will be explained.

(1) Core Particle

The core particle serves as a support on which a biologically activesubstance is to be immobilized. According to one embodiment of thedevice of the present invention (affinity particle), the “biologicallyactive substance” is for capturing a second biologically activesubstance in a sample by a specific bond of the biologically activesubstance and the second biologically active substance. Examples of thesample include body fluids such as blood, plasma and serum, cells suchas animal or plant cells and bacteria and so forth.

According to another embodiment of the device of the present invention,the biologically active substance acts as an agent used for therapeutictreatment (active ingredient) or as a ligand for bonding the agent.Examples of the biologically active substance include nucleic acids suchas DNA and RNA, proteins (including peptides) such as antigens,antibodies and enzymes, peptide nucleic acids, haptens, saccharides,glycopeptides and so forth. Among these, nucleic acids are preferred.The biologically active substance will be described later.

The aforementioned core particle is preferably insoluble in an aqueousmedium and preferably exhibits good dispersibility in an aqueous medium.Specific examples of the core particle include organic particles,inorganic particles or organic/inorganic composite particles made ofplastics, metals, carbon, natural polymers, ceramics (includinginorganic solids) and so forth.

Examples of the plastics include polyethylenes, polystyrenes,polycarbonates, polypropylenes, polyamides, phenol resins, epoxy resins,polycarbodiimide resins, polyvinyl chlorides, polyvinylidene fluorides,polyethylene fluorides, polyimides, acrylic resins and so forth.

Examples of the inorganic polymers include glass, quartz, carbon, silicagel, graphite and so forth.

Examples of the metals include metals existing as solids at an ordinarytemperature such as gold, platinum, silver, copper, iron, aluminum,magnet, paramagnet and apatite.

Examples of the natural polymers include cellulose, cellulosederivatives, chitin, chitosan, alginic acid and so forth.

Examples of the ceramics include alumina, silica, silicon oxide,aluminum hydroxide, magnesium hydroxide, silicon carbide, siliconnitride, boron carbide and so forth.

One kind of the aforementioned materials alone can be used, or two ormore kinds of them can be used in combination as a composite particle.

If the core particle is commercially available, it may be used.Alternatively, the core particle may be produced by any of various knownmethods. For example, if a desired particle is an organic particle or anorganic/inorganic composite particle, the following methods can be used.However, the methods are not particularly limited to these methods.

(i) A method of obtaining particles by pulverizing and classifying asolution resin obtained by usual bulk polymerization or solutionpolymerization.

(ii) A method of obtaining particles (including spherical particles) byadding dropwise a solution resin obtained by polymerization similar tothose mentioned above.

(iii) A method of obtaining particles (including spherical particles) byemulsification or suspension polymerization performed in an aqueoussolution.

(iv) A method of obtaining particles by employing the method of (iii) incombination with the seeding method or the like.

(v) A method of obtaining particles (mainly spherical particles) bydispersion polymerization in a non-aqueous solvent or a mixed solventwith water.

(vi) A method of obtaining particles by employing the method of (v) incombination with the seeding method or the like.

(vii) A method of obtaining pellet-like particles using an extruder orthe like.

Further, the particles obtained by the aforementioned polymerization mayoriginally have a crosslinked structure, and such particles can also beused for the production according to the present invention.

Preferably, a surface portion, or inside and surface portions of thecore particle desirably contain a compound (also referred to as“compound B” hereinafter) having a functional group that can bond to theorganic compound A described later by copolymerizing or mixing thecompound. For example, when the base particle is produced, the coreparticle may be modified beforehand, if necessary, for bonding of thecore particle and the organic compound A. Further, the organic compoundA may also be added to the core particle beforehand. The expression“modification” of the core particle includes both of the case where afunctional group is later introduced into a base material from which thecore particle is formed, and the case where a base material having afunctional group is produced by using a compound originally having afunctional group. The compound B will be explained later.

Various known methods can be adopted as the method of incorporating theaforementioned compound B into the core particle. Examples of suchmethods include, when the core particle is a polymer particle derivedfrom unsaturated monomers, a method of copolymerizing unsaturatedmonomers having a functional group that can bond to the organic compoundA during polymerization of the polymer to produce particles and soforth.

More specific examples include, when the particle to be used as a coreis a metal or an inorganic particle such as those of silicon oxide,aluminum hydroxide or magnesium hydroxide, a method of treating thesurface of the particle with a silane coupling agent having a functionalgroup that can bond to the organic compound A to form the core particleand so forth.

Further, when the particle to be used as a core is a composite particlecomprising organic and inorganic materials (polymer particles containinga magnetic substance etc.), for example, the core particle can also beproduced by employing the aforementioned methods in combinationdepending on the amounts of organic and inorganic material components.

The device of the present invention and the core particle serving as asupport therefor may have an irregular shape or spherical shapedepending of the use of the device. However, because highly precisedevices and particles with uniform surface areas have been required formedical use in recent years, particles having uniform particle diametersor particles of a spherical or substantially spherical shape arepreferred.

(2) Organic Compound A

The organic compound A is a compound having at least one functionalgroup A1 that can bond to the core particle and one functional group A2that can chemically bond to the biologically active substance as well astwo or more hydrophilic groups. The functional group A1 that can bond tothe core particle and the functional group A2 that can bond to thebiologically active substance may be identical or different.

Examples of the aforementioned functional groups A1 and A2 includecarbodiimide group, ester group, carbonate group, epoxy group, oxazolinegroup and so forth.

The aforementioned hydrophilic group is not particularly limited so longas it is a functional group that is swollen or dissolved in water.Specific examples thereof include hydroxyl group, n carboxyl group,ethylene oxide group, propylene oxide group, phosphoric acid group,sulfonic acid group, heterocyclic hydrophilic functional groupcontaining nitrogen and so forth. A molecule of the organic compound Apreferably contains two or more, preferably 8 or more, more preferably12 or more hydrophilic groups. Further, the upper limit number of thehydrophilic groups is usually 60 or less, preferably 60 or less, morepreferably 40 or less. The hydrophilic group is preferably a hydrophilicgroup that makes the organic compound A containing it water-soluble.

Among the aforementioned functional groups, carbodiimide group ispreferred, and in particular, a water-soluble carbodiimide compound ispreferred. Hereafter, the organic compound A containing carbodiimidegroup will be described with reference to examples thereof. The organiccompound A having carbodiimide group is preferably a compoundrepresented by the following formula.A_(x)—(R—X)_(n)—R—A_(y)  (I)

A_(x) and A_(y) independently represent a segment having a functionalgroup that exhibits hydrophilicity and may be identical to or differentfrom each other. R independently represents an organic group of two ormore valences, X independently represents carbodiimide group, epoxygroup or oxazoline group, and n is an integer of 2 to 80, preferably 2to 40.

Examples of the aforementioned organic group of two or more valencesinclude hydrocarbon groups, organic groups containing nitrogen atom oroxygen atom and so forth. Divalent hydrocarbon groups are preferred, andexamples thereof include C1 to C12 alkylene groups, C3 to C10cycloalkylene groups, C4 to C16 alkylene groups having a cyclic ornon-cyclic structure, C6 to C16 divalent aromatic rings, C7 to C18alkylene groups containing an aromatic ring and so forth.

Examples of the organic compound A having carbodiimide group andrepresented by the aforementioned formula (I) (also referred to simplyas “carbodiimide compound” hereinafter) include polycarbodiimides thatcan be produced by the method disclosed in Japanese Patent Laid-open No.51-61599, the method of L. M. Alberino et al. (J. Appl. Polym. Sci., 21,190 (1990)), the method disclosed in Japanese Patent Laid-open No.2-292316 or so forth. That is, those compounds can be produced fromorganic polyisocyanate compounds in the presence of a catalyst thatpromotes carbodiimidation of isocyanates.

Examples of the aforementioned organic polyisocyanate compounds used forthe production of polycarbodiimides include 4,4′-dicyclohexylmethanediisocyanate, m-tetramethylxylylene diisocyanate, 2,4-tolylenediisocyanate, 2,6-tolylene diisocyanate, a mixture of 2,4-tolylenediisocyanate and 2,6-tolylene diisocyanate, crude tolylene diisocyanate,crude methylene diphenyl diisocyanate, 4,4′,4″-triphenylmethylenetriisocyanate, xylene diisocyanate, hexamethylene-1,6-diisocyanate,lysine diisocyanate, hydrogenated methylene diphenyl diisocyanate,m-phenyl diisocyanate, naphthylene-1,5-diisocyanate, 4,4′-biphenylenediisocyanate, 4,4′-diphenylmethane diisocyanate,3,3′-dimethoxy-4,4′-biphenyl diisocyanate,3,3′-dimethyldiphenylmethane-4,4′-diisocyanate, isophorone diisocyanateand arbitrary mixtures thereof.

Carbodiimidation of isocyanate groups in the aforementionedpolyisocyanate compounds or mixtures thereof causes condensationpolymerization. This reaction is usually performed by heating anisocyanate in the presence of a carbodiimidation catalyst. In thisreaction, the molecular weight (degree of polymerization) of the productcan be controlled by adding a compound having a functional groupexhibiting reactivity with an isocyanate group, for example, hydroxylgroup, primary or secondary amino group, carboxyl group, thiol group orthe like as well as a hydrophilic functional group in the molecule as anend blocking agent at a suitable stage to block the end of thecarbodiimide compound. The degree of polymerization can also becontrolled by changing concentrations of polyisocyanate compounds or thelike and reaction time.

Examples of the aforementioned catalyst that promotes carbodiimidationof organic isocyanates include various substances, and1-phenyl-2-phospholene-1-oxide, 3-methyl-1-phenyl-2-phospholene-1-oxide,1-ethyl-2-phospholene-1-oxide, 3-phospholene isomers thereof and soforth are preferred in view of yield and other factors.

The carbodiimide compounds represented by the aforementioned chemicalformula (I) usually have an average molecular weight of 200 to 100,000,preferably 500 to 50,000.

As described above, to produce the carbodiimide compound according tothe present invention, the aforementioned isocyanate is first heated inthe presence of a carbodiimidation catalyst. In this case, the synthesismay be performed with or without a solvent. Further, a solvent may beadded during the process of the reaction. In such a case, the solventcan be suitably selected depending on the purpose of use.

Specifically, typical examples of the solvent include ketones such asacetone, methyl ethyl ketone, methyl isobutyl ketone and cyclohexanone;esters such as ethyl acetate, butyl acetate, ethyl propionate andcellosolve acetate; aliphatic or aromatic hydrocarbons such as pentane,2-methylbutane, n-hexane, cyclohexane, 2-methylpentane,2,2-dimethylbutane, 2,3-dimethylbutane, heptane, n-octane, isooctane,2,2,3-trimethylpentane, decane, nonane, cyclopentane,methylcyclopentane, methylcyclohexane, ethylcyclohexane, p-menthane,benzene, toluene, xylene and ethylbenzene; halogenated hydrocarbons suchas carbon tetrachloride, trichloroethylene, chlorobenzene andtetrabromoethane; ethers such as ethyl ether, dimethyl ether, trioxaneand tetrahydrofuran; acetals such as methylal and diethylacetal; organiccompounds containing sulfur or nitrogen such as nitropropene,nitrobenzene, pyridine, dimethylformamide and dimethyl sulfoxide and soforth. The solvent is not particularly limited so long as it does notadversely affect the isocyanate group and the carbodiimide group at thetime of the synthesis, and the solvent can be suitably selecteddepending on the purpose of the polymerization. Further, one kind ofthese solvents alone can be used, or two or more kinds of them may beused in combination.

Further, if the carbodiimide resin ends are blocked with ahydrophilizing segment described below after completion of thesynthesis, water, alcohols such as methanol, ethanol, 1-propanol,2-propanol, 1-butanol, 2-butanol, isobutyl alcohol, tert-butyl alcohol,1-pentanol, 2-pentanol, 3-pentanol, 2-methyl-1-butanol, isopentylalcohol, tert-pentyl alcohol, 1-hexanol, 2-methyl-1-pentanol,4-methyl-2-pentanol, 2-ethylbutanol, 1-heptanol, 2-heptanol, 3-heptanol,2-octanol, 2-ethyl-1-hexanol, benzyl alcohol and cyclohexanol; etheralcohols such as methyl cellosolve, ethyl cellosolve, isopropylcellosolve, butyl cellosolve and diethylene glycol monobutyl ether andso forth can be used as a diluent in addition to the aforementionedsolvents. One kind of these alone can be used, or two or more kinds ofthem may be used in combination. It is preferable to use a relativelylow temperature during the dilution, because carbodiimide group ishighly reactive.

By changing molecular weight or composition of the organic compound Asuch as those carbodiimide compounds or the hydrophilizing segment,dispersibility of the base particle can be freely controlled, anddegrees of aggregation and dispersion of the device itself can becontrolled as required.

The hydrophilizing segment (A_(x) and A_(y) in the aforementionedformula) is not particularly limited so long as it is, for example, asegment that has a hydrophilic group and can become water-soluble.Preferred examples thereof include residues of alkylsulfonates having atleast one of reactive hydroxyl group such as sodiumhydroxyethanesulfonate and sodium hydroxypropanesulfonate, quaternarysalts of residues of dialkylaminoalcohols such as2-dimethylaminoethanol, 2-diethylaminoethanol,3-dimethylamino-1-propanol, 3-diethylamino-1-propanol,3-diethylamino-2-propanol, 5-diethylamino-2-propanol and2-(di-n-butylamino)ethanol, quaternary salts of residues ofdialkylaminoalkylamines such as 3-dimethylamino-n-propylamine,3-diethylamino-n-propylamine and 2-(diethylamino)ethylamine and residuesof poly(alkylene oxides) having at least one of reactive hydroxyl groupsuch as poly(ethylene oxide) monomethyl ether, poly(ethylene oxide)monoethyl ether, poly(ethylene oxide/propylene oxide) monomethyl etherand poly(ethylene oxide/propylene oxide) monoethyl ether. One kind ofthese segments (A_(x) and A_(y)) that become hydrophilic alone can beused, or two or more kinds of them may be used in combination, and theycan also be used as copolymerized mixed compounds.

Because devices used for medical test, diagnosis or therapeutictreatment, in particular, are often used with water-soluble mediaincluding water, dispersibility of the base particle greatly affect theprecision of the device.

For the production of the base particle, one kind of the aforementionedorganic compounds A alone can be used, or two or more kinds of them maybe used in combination. They may also be used as copolymerized mixedcompounds.

(3) Base Particle

The base particle consists of the aforementioned core particle bondedwith the organic compound A by a chemical bond. In the presentinvention, the “chemical bond” means a bond such as covalent bond,coordinate bond or ionic bond.

As for the production method of the base particle, the base particle canbe obtained by, for example, preparing a core particle containing acompound B having a functional group that can react with an organiccompound A, adding the organic compound A to the core particle in thepresence of a solvent in which the core particle is not dissolved andthe organic compound A is dissolved to allow a chemical reaction,without deforming the shape of the particle. In the production, if thecore particle and the organic compound A are not chemically bonded inthe base particle, impurities or undesired substances are oftendissolved or precipitated in the solution in the following processes, orthe particles often aggregate. As a result, the obtained device can nolonger maintain high precision required as a device for test, diagnosisor therapeutic treatment.

Hereafter, the method of producing the base particle in which a coreparticle and an organic compound A are bonded will be explained byreferring to a case where polymer particles are used as the coreparticle as an example.

Examples of the polymer particles that can be used for the core particleinclude, for example, those of styrene polymers, (meth)acrylic polymers,copolymers obtained by addition polymerization of other vinyl polymers,polymers obtained by hydrogen transfer polymerization, polymers obtainedby polycondensation, polymers obtained by addition condensation and soforth.

Typical examples of copolymerizable raw material monomers as the maincomponent include (i) styrenes such as styrene, o-methylstyrene,m-methylstyrene, p-methylstyrene, α-methylstyrene, p-ethylstyrene,2,4-dimethylstyrene, p-n-butylstyrene, p-tert-butylstyrene,p-n-hexylstyrene, p-n-octylstyrene, p-n-nonylstyrene, p-n-decylstyrene,p-n-dodecylstyrene, p-methoxystyrene, p-phenylstyrene, p-chlorostyreneand 3,4-dichlorostyrene, (ii) (meth)acrylic acid esters such as methylacrylate, ethyl acrylate, n-butyl acrylate, isobutyl acrylate, propylacrylate, hexyl acrylate, 2-ethylhexyl acrylate, n-octyl acrylate,dodecyl acrylate, lauryl acrylate, stearyl acrylate, 2-chloroethylacrylate, phenyl acrylate, methyl α-chloroacrylate, methyl methacrylate,ethyl methacrylate, n-butyl methacrylate, isobutyl methacrylate, propylmethacrylate, hexyl methacrylate, 2-ethylhexyl methacrylate, n-octylmethacrylate, dodecyl methacrylate, lauryl methacrylate and stearylmethacrylate, (iii) vinyl esters such as vinyl acetate, vinylpropionate, vinyl benzoate and vinyl butyrate, (iv) (meth)acrylic acidderivatives such as acrylonitriles and methacrylonitriles, (v) vinylethers such as vinyl methyl ether, vinyl ethyl ether and vinyl isobutylether, (vi) vinyl ketones such as vinyl methyl ketone, vinyl hexylketone and methyl isopropenyl ketone, (vii) N-vinyl compounds such asN-vinylpyrrole, N-vinylcarbazole, N-vinylindole and N-vinylpyrrolidone,(viii) vinyl fluoride, vinylidene fluoride, tetrafluoroethylene,hexafluoropropylene, (meth)acrylic esters having a fluorinated alkylgroup such as trifluoroethyl acrylate and tetrafluoropropyl acrylate andso forth. One kind of these alone can be used, or two or more kinds ofthem may be used in combination.

It is sufficient that the core particle in the base particle accordingto the present invention should contain a compound B containing afunctional group B1 that can bond to an organic compound A in a surfaceportion or inside and surface portions thereof. Examples of theaforementioned functional group B1 include, for example, compoundshaving groups containing a carbon-carbon unsaturated bond (double bond,triple bond), α,β-unsaturated carbonyl group, epoxy group, isocyanategroup, carboxyl group, hydroxyl group, amido group, thiol group, cyanogroup, amino group, chloromethyl group, glycidyl ether group, estergroup, formyl group, nitrile group, nitroso group, carbodiimide group,oxazoline group or the like. One kind of these alone can be used, or twoor more kinds of them may be used in combination. Carboxyl group,hydroxyl group, primary or secondary amino group or thiol group arepreferred.

Further, specific examples of the compound B include radicallypolymerized monomers and compounds containing carboxyl group. Typicalexamples thereof include various unsaturated mono- or dicarboxylic acidsor unsaturated dibasic acids such as acrylic acid, methacrylic acid,crotonic acid, itaconic acid, maleic acid, fumaric acid, monobutylitaconate and monobutyl maleate and so forth. One kind of thesecompounds alone can be used, or two or more kinds of them may be used incombination.

Examples of the compound B further include radically polymerizedmonomers and compounds having hydroxyl group. Typical examples thereofinclude (meth)acrylic monomers such as 2-hydroxyethyl (meth)acrylate,2-hydroxypropyl (meth)acrylate, 3-hydroxypropyl (meth)acrylate and4-hydroxybutyl (meth)acrylate, polyalkylene glycol (meth)acrylatecompounds such as polyethylene glycol mono(meth)acrylate andpolypropylene glycol mono(meth)acrylate, various hydroxyalkyl vinylethers such as hydroxyethyl vinyl ether and hydroxybutyl vinyl ether,various allyl compounds such as allyl alcohol and 2-hydroxyethyl allylether and so forth. One kind of these compounds alone can be used, ortwo or more kinds of them may be used in combination.

Examples of the compound B further include polymers containing hydroxylgroup. Typical examples thereof include thermoplastic resins containinghydroxyl group such as completely or partially saponified resins ofpolyvinyl alcohol (PVA) and saponified resins of polymers containingacetic acid ester comprising a copolymer of vinyl acetate and othervinyl monomers. One kind of these compounds alone may be used, or two ormore kinds of them may be used in combination.

Examples of the compound B further include radically polymerizedmonomers and compounds containing amino group. Typical examples thereofinclude, specifically, derivatives of alkyl esters of acrylic ormethacrylic acids such as aminoethyl acrylate, N-propylaminoethylacrylate, N-ethylaminopropyl methacrylate, N-phenylaminoethylmethacrylate and N-cyclohexylaminoethyl methacrylate; allylamine andallyl amine derivatives such as N-methylallylamine; styrene derivativessuch as p-aminostyrene; triazine derivatives such as2-vinyl-4,6-diamino-S-triazine and so forth, and compounds containingprimary or secondary amino group are preferred. One kind of thesecompounds alone may be used, or two or more kinds of them may be used incombination.

Examples of the compound B further include radically polymerizedmonomers and compounds containing thiol (mercapto) group. Typicalexamples thereof include, specifically, monomers or compounds containingmercapto (thiol) group and having an unsaturated double bond such as2-propene-1-thiol, 3-butene-1-thiol, 4-pentene-1-thiol, 2-mercaptoethyl(meth)acrylate, 2-mercapto-1-carboxyethyl (meth)acrylate,N-(2-mercaptoethyl)acrylamide, N-(2-mercapto-1-carboxyethyl)acrylamide,N-(2-mercaptoethyl)methacrylamide, N-(4-mercaptophenyl)acrylamide,N-(7-mercaptonaphthyl)acrylamide and mono-2-mercaptoethylamide maleateand so forth. One kind of these compounds alone may be used, or two ormore kinds of them may be used in combination. Examples further includethermoplastic resins having thiol (mercapto) group such as modifiedpolyvinyl alcohols having thiol (mercapto) group and so forth.

Further, when a composite group of carboxyl group, hydroxyl group, aminogroup, thiol (mercapto) group etc. is desired to be introduced into acopolymer that forms the core particle, a polyfunctional copolymer canbe produced by using two or more kinds of monomers containing any of theaforementioned various reactive groups in combination.

A polyfunctional base particle can be produced by controlling theamounts of the aforementioned functional groups, amount of the organiccompound A to be added, reaction temperature and other conditions duringthe reaction of the core particle and the organic compound A.

As a polymerization initiator used in the polymerization of radicallypolymerizable monomers for the production of the core particle, knownradical polymerization initiators can be used. Typical examples thereofinclude, specifically, peroxides such as benzoyl peroxide, cumenehydroperoxide, t-butyl hydroperoxide, sodium persulfate and ammoniumpersulfate, azo compounds such as azobisisobutyronitrile,azobismethylbutyronitrile and azobisvaleronitrile and so forth. One kindof these compounds alone may be used, or two or more kinds of them maybe used in combination.

For the production of the core particle, various synthesis andpolymerization methods such as those mentioned above are used. Examplesinclude not only synthesis without solvent such as bulk polymerizationbut also synthesis in a solvent such as solution polymerization.Specific examples of the polymerization solvent include water, alcoholssuch as methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol,isobutyl alcohol, tert-butyl alcohol, 1-pentanol, 2-pentanol,3-pentanol, 2-methyl-1-butanol, isopentyl alcohol, tert-pentyl alcohol,1-hexanol, 2-methyl-1-pentanol, 4-methyl-2-pentanol, 2-ethylbutanol,1-heptanol, 2-heptanol, 3-heptanol, 2-octanol, 2-ethyl-1-hexanol, benzylalcohol and cyclohexanol; ether alcohols such as methyl cellosolve,ethyl cellosolve, isopropyl cellosolve, butyl cellosolve and diethyleneglycol monobutyl ether; ketones such as acetone, methyl ethyl ketone,methyl isobutyl ketone and cyclohexanone; esters such as ethyl acetate,butyl acetate, ethyl propionate and cellosolve acetate; aliphatic oraromatic hydrocarbons such as pentane, 2-methylbutane, n-hexane,cyclohexane, 2-methylpentane, 2,2-dimethylbutane, 2,3-dimethylbutane,heptane, n-octane, isooctane, 2,2,3-trimethylpentane, decane, nonane,cyclopentane, methylcyclopentane, methylcyclohexane, ethylcyclohexane,p-menthane, dicyclohexyl, benzene, toluene, xylene and ethylbenzene;halogenated hydrocarbons such as carbon tetrachloride,trichloroethylene, chlorobenzene and tetrabromoethane; ethers such asethyl ether, dimethyl ether, trioxane and tetrahydrofuran; acetals suchas methylal and diethylacetal; fatty acids such as formic acid, aceticacid and propionic acid; organic compounds containing sulfur or nitrogensuch as nitropropene, nitrobenzene, dimethylamine, monoethanolamine,pyridine, dimethylformamide, dimethyl sulfoxide and acetonitrile and soforth. The polymerization solvent is not particularly limited and may besuitably selected depending on the purpose of use of the polymerizationmethod. One kind of these solvents alone may be used, or two or morekinds of them may be used in combination.

Further, in the production of the core particle, a dispersing agent,stabilizer, emulsifier (or surfactant), antioxidant, catalyst (orreaction accelerator) and so forth may be suitably used depending on thepolymerization method that can be used.

Typical examples of the dispersing agent and the stabilizer include,specifically, various hydrophobic or hydrophilic dispersing agents andstabilizers, for example, polystyrene derivatives such aspolyhydroxystyrene, polystyrenesulfonic acid, vinylphenol/(meth)acrylicacid ester copolymer, styrene/(meth)acrylic acid ester copolymers andstyrene/vinylphenol/(meth)acrylic acid ester copolymers;poly(meth)acrylic acid derivatives such as poly(meth)acrylic acid,poly(meth)acrylamide, polyacrylonitrile, polyethyl (meth)acrylate andpolybutyl (meth)acrylate; polyvinyl alkyl ether derivatives such aspolymethyl vinyl ether, polyethyl vinyl ether, polybutyl vinyl ether andpolyisobutyl vinyl ether; cellulose derivatives such as cellulose,methylcellulose, cellulose acetate, cellulose nitrate,hydroxymethylcellulose, hydroxyethylcellulose, hydroxypropylcelluloseand carboxymethylcellulose; polyvinyl acetate derivatives such aspolyvinyl alcohol, polyvinyl butyral, polyvinyl formal and polyvinylacetate; nitrogen-containing polymer derivatives such aspolyvinylpyridine, polyvinylpyrrolidone, polyethylenimine andpoly-2-methyl-2-oxazoline; poly(halogenated vinyl derivatives) such aspolyvinyl chloride and polyvinylidene chloride; polysiloxane derivativessuch as polydimethylsiloxane and so forth. One kind of these compoundsalone may be used, or two or more kinds of them may be used incombination.

Examples of the emulsifier (surfactant) include anionic emulsifiers, forexample, alkylsulfuric ester salts such as sodium laurylsulfate,alkylbenzenesulfonic acid salts such as sodium dodecylbenzenesulfonate,alkylnaphthalenesulfonic acid salts, fatty acid salts, alkylphosphoricacid salts and alkylsulfosuccinic acid salts; cationic emulsifiers suchas alkylamine salts, quaternary ammonium salts, alkylbetaines and amineoxides; nonionic emulsifiers such as polyoxyethylene alkyl ethers,polyoxyethylene alkyl ethers, polyoxyethylene alkylallyl ethers,polyoxyethylene alkylphenyl ethers, sorbitan fatty acid esters, glycerinfatty acid esters and polyoxyethylene fatty acid esters and so forth.One kind of these compounds alone may be used, or two or more kinds ofthem may be used in combination.

Further, in the production of the core particle, a small amount of acrosslinking agent may be added depending on the purpose of use. Typicalexamples thereof include, specifically, aromatic divinyl compounds suchas divinylbenzene and divinylnaphthalene; and other compounds such asethylene glycol diacrylate, ethylene glycol dimethacrylate, triethyleneglycol dimethacrylate, tetraethylene glycol dimethacrylate, 1,3-butyleneglycol dimethacrylate, trimethylolpropane triacrylate,trimethylolpropane trimethacrylate, 1,4-butanediol diacrylate, neopentylglycol diacrylate, 1,6-hexanediol diacrylate, pentaerythritoltriacrylate, pentaerythritol tetraacrylate, pentaerythritoldimethacrylate, pentaerythritol tetramethacrylate, glycerolacroxydimethacrylate, N,N-divinylaniline, divinyl ether, divinyl sulfideand divinyl sulfone. One kind of these compounds alone may be used, ortwo or more kinds of them may be used in combination.

When the core particle is a thermoplastic particle containing organicsubstances, only the particle surface (surface layer portion) or insideand surface portions may be crosslinked.

Examples of the antioxidant include phenol antioxidants, sulfurantioxidants, phosphorus antioxidants, amine antioxidants, hydroquinoneantioxidants, hydroxylamine antioxidants and so forth.

Further, the catalyst (reaction accelerator) is not particularly limitedso long it accelerates the reaction, and known catalysts may be used.The catalyst may be suitably selected so that physical properties of theparticle should not be adversely affected, and a suitable amount thereofcan be added. For example, when at least one of the functional group ofthe core particle and the functional group of the organic compound Acontains epoxy group, a catalyst selected from, specifically, tertiaryamines such as benzyldimethylamine, triethylamine, tributylamine,pyridine and triphenylamine; quaternary ammonium compounds such astriethylbenzylammonium chloride and tetramethylammonium chloride;phosphines such as triphenylphosphine and tricyclophosphine; phosphoniumcompounds such as benzyltrimethylphosphonium chloride; imidazolecompounds such as 2-methylimidazole and 2-methyl-4-ethylimidazole;alkaline metal hydroxides such as potassium hydroxide, sodium hydroxideand lithium hydroxide; alkaline metal carbonates such as sodiumcarbonate and lithium carbonate; alkaline metal salts of organic acids;halogenides exhibiting properties of Lewis acid such as borontrichloride, boron trifluoride, tin tetrachloride and titaniumtetrachloride and complex salts thereof and so forth can be added. Onekind of these compounds alone may be used, or two or more kinds of themmay be used in combination.

When the aforementioned core particle is a particle containing organicsubstances, the weight average molecular weight is about 1000 to3,000,000. When the core particle is a spherical particle, the weightaverage molecular weight is about 3000 to 1,000,000.

The amount of the core particle having functional groups with which theorganic compound A can react, which is used for the production of thebase particle, preferably corresponds to, when the core particle is apolymer microparticle, 30 to 2000 equivalents, more preferably 50 to1000 equivalents, further preferably 80 to 900 equivalents, particularlypreferably 100 to 500 equivalents, with respect to the content of thefunctional group. If the amount exceeds 2000 equivalents, the bonding tothe core particle requires considerable time because the amount of thefunctional groups becomes too small, and thus such an amount may not bepreferred. On the other hand, if the amount is less than 30 equivalents,the bonding density becomes too high, and functional groups that canbond to a biologically active substance may not be left on the surfaceand the surface layer portion of the base particle. However, if there isextra time or a minimal amount of the functional groups is required, theamount may not be within the range defined above. That is, the amount ofthe core particle may be more than 2000 equivalents per functional groupor less than 30 equivalents per functional group.

This also applies to the cases where the core particle is an inorganicparticle, organic/inorganic composite particle or the like.

The aforementioned term “equivalent” means a certain amount assigned toeach compound on the basis of quantitative relations of substances in achemical reaction. For example, the equivalent of the core particle ofthe present invention means the chemical formula weight of the coreparticle per mole of the functional group that can react with theorganic compound A.

The amount of the organic compound A required to produce the baseparticle is 50 to 1500 equivalents, preferably 80 to 1000 equivalents,more preferably 100 to 800 equivalents, particularly preferably 200 to700 equivalents, with respect to the functional group. If the amountexceeds 1500 equivalents, the bonding to the core particle requiresconsiderable time because the amount of the functional group becomes toosmall, and thus such an amount may not be preferred. On the other hand,if the amount is less than 50 equivalents, a lot of functional groupsthat can bond to a biologically active substance are remained, and theymay provide bad influences. However, if there is extra time or a minimalamount of the functional groups is required, the amount may not bewithin the range defined above. That is, the amount may be more than1500 equivalents per functional group.

In the production of the base particle, although the amount of theorganic compound A to be added to the core particle depends on therequired amount of residual organic compounds after curing or bonding,the organic compound A may be added in an amount of about 0.1 to 20,preferably 0.5 to 8, more preferably 1 to 5, in terms of the equivalentratio with respect to the functional group of the core particle. This isalso applicable to the cases where the particle to be used as the coreis a core particle made of an organic/inorganic composite particle or aninorganic particle. Further, the addition amount of the organic compoundA may exceed 20 in terms of the equivalent ratio. However, such anamount results in a large amount of residual organic compounds in themedium and thus is not preferred in view of cost. Further, if theaddition amount is less than 0.1 in terms of the equivalent ratio, thefunctional group that can bond to a biologically active substance maynot be remained. However, if there is extra time or a minimal amount ofthe functional groups is required, the amount may not be within therange defined above.

The average particle diameter of the base particle is preferably 0.01 to100 μm, more preferably 0.05 to 50 μm, further preferably 0.08 to 30 μm,particularly preferably 0.1 to 10 μm. If the average particle diameterexceeds 100 μm, the precipitation rate of the particle increases, andthis is not preferable as a device for biological or medical use. On theother hand, if the average particle diameter is less than 0.01 μm, thedegree of aggregation becomes high because the particle diameter is toosmall, and monodispersed particles may not be obtained. It is preferablethat diameters of 80% or more, preferably 90% or more, more preferably95% or more, of the base particles satisfy the aforementioned range.

Although the reaction temperature of the reaction for obtaining the baseparticle depends on the type of the solvent, it is preferably within therange of 10 to 200° C., more preferably 15 to 150° C., furtherpreferably 20 to 130° C.

Further, the reaction time may be time required to almost complete thebonding reaction of the core particle and the functional group of theorganic compound A. Although it largely depends on the type and amountof the organic compound used, the type of the functional group in theparticle, viscosity and concentration of the solution and so forth, itis, for example, about 1 to 24 hours, preferably 2 to 15 hours, at 50°C. The base particle can be obtained even if the aforementioned factorsare changed to extend the reaction time (longer than 24 hours). However,prolonged time may not be preferred in view of production method.Preferred reaction time can be easily determined by performing thereaction using various reaction times in preliminary experiments.

The solvent in which the core particle is not dissolved and the organiccompound A is dissolved is at least one kind of solvent selected fromwater and organic solvents, and may be suitably selected considering thetype and amount of the organic compound used, type of the resin to beused as a component of the base particle, the type of the containedfunctional group, the purpose of use and so forth.

Specific examples of the solvent include water, alcohols such asmethanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol,isobutyl alcohol, tert-butyl alcohols, 1-pentanol, 2-pentanol,3-pentanol, 2-methyl-1-butanol, isopentyl alcohol, tert-pentyl alcohols,1-hexanol, 2-methyl-1-pentanol, 4-methyl-2-pentanol, 2-ethylbutanol,1-heptanol, 2-heptanol, 3-heptanol, 2-octanol, 2-ethyl-1-hexanol, benzylalcohol and cyclohexanol; ether alcohols such as methyl cellosolve,ethyl cellosolve, isopropyl cellosolve, butyl cellosolve and diethyleneglycol monobutyl ether; ketones such as acetone, methyl ethyl ketone,methyl isobutyl ketone and cyclohexanone; esters such as ethyl acetate,butyl acetate, ethyl propionate and cellosolve acetate; aliphatic oraromatic hydrocarbons such as pentane, 2-methylbutane, n-hexane,cyclohexane, 2-methylpentane, 2,2-dimethylbutane, 2,3-dimethylbutane,heptane, n-octane, isooctane, 2,2,3-trimethylpentane, decane, nonane,cyclopentane, methylcyclopentane, methylcyclohexane, ethylcyclohexane,p-menthane, dicyclohexyl, benzene, toluene, xylene and ethylbenzene;halogenated hydrocarbons such as carbon tetrachloride,trichloroethylene, chlorobenzene and tetrabromoethane; ethers such asethyl ether, dimethyl ether, trioxane and tetrahydrofuran; acetals suchas methylal and diethylacetal; fatty acids such as formic acid, aceticacid and propionic acid; organic compounds containing sulfur or nitrogensuch as nitropropene, nitrobenzene, dimethylamine, monoethanolamine,pyridine, dimethylformamide, dimethyl sulfoxide and acetonitrile and soforth. Preferred examples include water-soluble or hydrophilic mediaincluding water, lower alcohols such as methanol and ethanol, etheralcohols such as methyl cellosolve and ethyl cellosolve, mixtures ofwater and a lower alcohol and mixtures of water and an ether alcohol,toluene, dimethylformamide (DMF), tetrahydrofuran (THF), methyl ethylketone (MEK), methyl isobutyl ketone (MIBK), acetone,N-methyl-2-pyrrolidone (NMP), dichloromethane, tetrachloroethylene andso forth. Further preferred are water-soluble or hydrophilic mediaincluding water, lower alcohols such as methanol and ethanol, mixturesof water and a lower alcohol such as methanol and ethanol, mixtures ofwater and a lower alcohol such as methanol and ethanol, and mixtures ofwater and an ether alcohol. These solvents are not particularly limited,and a solvent suitable for the purpose of use may be selected asrequired. One kind of these solvents alone may be used, or two or morekinds of them may be used in combination.

In the production of the base particle, the aforementioned dispersingagents, antioxidants, stabilizers, emulsifiers (surfactants), catalysts(reaction accelerators) and so forth can also be suitably selected andadded as required.

In the production of the base particle, the solution concentration usedfor the reaction of the core particle and the organic compound A is 1 to60% by weight, preferably 5 to 40% by weight, more preferably 10 to 30%by weight, as calculated according to the following equation.Solution concentration(% by weight)=[(Total solution−Solvent)/Totalsolution]×100

If the aforementioned solution concentration exceeds 80% by weight, theamount of the core particle or the organic compound A becomes excessive,therefore balance in the solution is deteriorated, and it becomesdifficult to obtain stable monodispersed particles. Therefore, such aconcentration is not preferred. Further, if the aforementioned solutionconcentration is less than 1% by weight, although the base particle canbe produced, synthesis needs to be performed over a long period of timeto obtain objective particles. In view of production, it is notdesirable to be required a long period of time.

As described above, as for the shape of the base particle, the particlespreferably have uniform particle diameters and have a spherical orsubstantially spherical shape. In the present invention, the “sphericalor substantially spherical” shape is defined as a shape satisfying thecondition of “1≦Major axis/Minor axis≦1.2” in a two-dimensionalprojection drawing of the particle. The major axis and the minor axiscan be measured as follows, for example. Particles are photographed byusing a scanning electron microscope (hereinafter referred to as “SEM”,e.g., Hitachi S-2150) at a measurable magnification (×100 to 10,000),and the diameter of one particle is randomly measured 15 times tomeasure the major axis and the minor axis. This procedure is randomlyrepeated (for example, n=100) to measure them.

Further, the average particle diameter can be obtained by photographingthe particles using SEM with a measurable magnification (×100 to10,000), randomly measuring diameters of particles (for example, n₁=500particles) and calculating the average of the diameters as the averageparticle diameter.

Further, from the measurement results of the aforementioned particlediameters of the core particles and the base particles, the CV(coefficient of variation) value for the particle diameter distributionas defined by the following equation can be obtained to confirmdistribution accuracy for each of the core particle, the base particleand the device.CV(%)=(Standard deviation of particle[device]diameter/Averageparticle[device]diameter)×100

Further, dispersibility of the base particle and the device of thepresent invention can be represented by the CV ratios defined as thefollowing equations. It is preferred that, among these CV ratios, atleast one of the CV_(b) ratio and the CV_(c) ratio is 0.6 to 3.0,preferably 0.8 to 1.5, more preferably 0.9 to 1.1. Further, it is morepreferred that both of the aforementioned CV_(b) ratio and CV_(c) ratioare within the aforementioned range. Further, it is particularlypreferred that the CV_(a) ratio is also within the aforementioned rangesin addition to the CV_(b) ratio and the CV_(c) ratio.CV _(a)ratio=CV ₁ /CV ₂CV _(b)ratio=CV ₁ /CV ₃CV _(c)ratio=CV ₂ /CV ₃CV ₁=(Standard deviation of core particle diameter/Average core particlediameter)×100CV ₂=(Standard deviation of base particle diameter/Average base particlediameter)×100CV ₃=(Standard deviation of device diameter/Average device particlediameter)×100(4) Biologically Active Substance

The biologically active substance is for capturing a second biologicallyactive substance that specifically bonds to the substance. Examples ofthe second biologically active substance to be detected include nucleicacids, proteins (including peptides), saccharides and so forth. Amongthem, nucleic acids are preferred. Further, when the device of thepresent invention is used for therapeutic treatment, the biologicallyactive substance functions as an active ingredient of an agent fortherapeutic treatment.

When a nucleic acid is used as the biologically active substance, it maynot be particularly different from nucleic acids used for usualhybridization of nucleic acids using nucleic acids immobilized on asolid phase, and it is not particularly limited so long as it is anucleic acid that can hybridize. Examples include, for example,naturally occurring and synthesized DNAs (including oligonucleotides)and RNAs (including oligonucleotides). Further, the nucleic acid may besingle-stranded or double-stranded. The chain length of the nucleic acidis not particularly limited so long as it allows hybridization. However,it is usually about 5 to 50,000 nucleotides, preferably 20 to 10,000nucleotides. Further, the nucleic acid may have a polymer containing agroup that becomes reactive with an ultraviolet ray such as thymine atthe 5′ or 3′ end.

Hereafter, a method of bonding a nucleic acid as the biologically activesubstance to the base particle will be exemplified. When othersubstances are used, the solvent, reaction conditions and so forth canbe suitably selected depending on the type of the functional group A2that can covalently bond to the biologically active substance in theorganic compound A, so that a reaction of forming a covalent bondbetween the biologically active substance and the functional group Ashould occur.

The solvent for dissolving a nucleic acid is not particularly limitedeither, and examples thereof include distilled water and buffers usuallyused for preparation of a nucleic acid solution, for example, Trisbuffers such as TE buffer (10 mM Tris/hydrochloric acid, pH 8.0/1 mMEDTA), aqueous solutions containing sodium chloride, aqueous solutionscontaining a carboxylate (sodium citrate, ammonium citrate, sodiumacetate etc.), aqueous solutions containing a sulfonate (sodiumdodecylsulfate, ammonium dodecylsulfate etc.), aqueous solutionscontaining a phosphonate (sodium phosphate, ammonium phosphate etc.) andso forth. Commercially available solvents such as Micro SpottingSolution (TeleChem International, Inc.) etc. can also be mentioned.Further, although the concentration of the nucleic acid solution is notparticularly limited either, the concentration is usually 1 mmol/ml to 1fmol/ml, preferably 100 pmol/ml to 100 fmol/ml.

Examples of the method of bringing a nucleic acid solution into contactwith the base particle include a method of adding a nucleic acidsolution dropwise onto base particles using a pipette, a method of usinga commercially available spotter, a method of suspending base particlesin a nucleic acid solution and so forth. Although the amount of thenucleic acid solution is not particularly limited, it is preferably 10nl to 10 ml. One kind or two or more kinds of nucleic acid solutions canbe used. As a positive control for confirming immobilization of thenucleic acid on the base particle, a labeled nucleic acid may be broughtinto contact with the base particle.

In a preferred embodiment of the present invention, a nucleic acidsolution is brought into contact with base particles and irradiated withultraviolet ray. Further, after the aforementioned nucleic acid solutionis brought into contact, the base particles can be dried beforeultraviolet ray irradiation. The aforementioned nucleic acid solutionmay be dried spontaneously or by heating. The temperature for theheating is usually 30 to 100° C., preferably 35 to 45° C.

Subsequently, the base particles are irradiated with an ultraviolet ray.Specifically, the ultraviolet ray may have a broad waveform including awavelength of 280 nm. The irradiation dose is usually 100 mJ/cm² ormore, preferably 200 mJ/cm² or more, as a cumulative irradiation dose.Further, a nucleic acid having a photoreactive group introduced into anarbitrary part of the nucleic acid may also be used.

The device of the present invention is produced by immobilizing anucleic acid onto a base particle as described above. The deviceobtained by the present invention can be used for, for example, analysisof nucleic acids by hybridization. Because a nucleic acid immobilized onbase particles by the method of the present invention exhibits superiordispersibility, more favorable detection sensitivity and reproducibilitycan be obtained compared with conventional methods. Hybridization anddetection thereof can be performed in the same manner as usualhybridization using nucleic acids immobilized on a solid phase.

Further, when a protein is used as the biologically active substance,any of proteins such as antibodies, antigens, enzymes and hormones canbe used as in usual solid phase immunological reagents.

<2> Utilization of Device of the Present Invention

In a preferred embodiment, the device of the present invention is usedfor detection or measurement of a second biologically active substancethat specifically bonds to a biologically active substance on thedevice. In this embodiment, the device of the present invention can alsodetect or measure a substance that inhibits the binding of thebiologically active substance on the device and the second biologicalsubstance.

Examples of the aforementioned biologically active substance includenucleic acids, proteins (including peptides), saccharides and so forth.Examples of nucleic acids include DNAs and RNAs, and examples ofproteins include antigens, antibodies, enzymes and so forth. Thefollowing explanation will be made for a nucleic acid as an example ofthe biologically active substance. However, except that a hybrid isformed as a detection method unique to nucleic acids, methods andconditions usually used for detection can also be adopted for othersubstances.

The device of the present invention can be used for detection orpurification or as a template for PCR in methods for detecting a nucleicacid by hybridization using a nucleic acid labeled with a labelingsubstance. That is, a particle comprising a base particle on which anucleic acid is immobilized (hereinafter, referred to as “device”) canbe used as a probe for hybridization.

A nucleic acid to be measured can be detected by hybridizing a probewith the nucleic acid to be measured to form a nucleic acid/nucleic acidhybrid, removing free probes from the system and detecting the labelingsubstance contained in the hybrid. Further, an objective nucleic acidcan also be purified in a similar manner. Alternatively, a nucleic acidcaptured by the nucleic acid immobilized on the device can be used as atemplate for PCR.

In the present invention, the base particle can be directly detected bymeasuring fluorescence intensity or the like using a fluorescencespectrophotometer, fluorescence spectrophotometer for a 96-wellmicrotiter plate, fluorescence microscope or the like.

Hybridization using the device of the present invention is notparticularly different from usual hybridization of nucleic acids.

Although a nucleic acid used as a sample is preferably labeled bylabeling a polynucleotide or oligonucleotide using a method usually usedfor labeling of a nucleic acid, a nucleic acid can also be labeled byincorporating a labeled nucleotide into a polynucleotide oroligonucleotide using a polymerase reaction.

The device of the present invention shows favorable dispersion stabilityin an aqueous medium, because the biologically active substance bonds tothe base particle via an organic compound having two or more hydrophilicgroups. For example, when detection of SNP (Single nucleotidepolymorphism) associated with a disease or gene expression analysis isperformed by hybridization, conventional devices aggregate with oneanother in a hybridization solution, and hence devices become unable tokeep an appropriate distance between them. As a result, hybridization iseasily inhibited by steric hindrance of devices or genes. On the otherhand, because of the favorable dispersibility of the devices of thepresent invention in an aqueous medium, the aforementioned inhibition ofhybridization hardly occurs. Further, due to this superiordispersibility in the aqueous solution, detection of specimen usingfluorescence, radioisotope or the like can be performed with goodreproducibility. As a result, SNP can be detected or gene expressionanalysis can be performed with high efficiency and sensitivity. Inparticular, when a biological sample is detected, it is often the casethat only a minimal amount of specimen can be collected. Accordingly,establishment of a technique for detecting a small amount of a substancewith high efficiency and sensitivity is being required. Because adetection method using the device of the present invention can detectsuch a minimal amount of specimen with good reproducibility, it would bean effective detection technique.

In the present invention, an aqueous medium means any of water, bufferssuch as TE buffer, SSC buffer, phosphate buffer, acetate buffer, boratebuffer, Tris-HCl buffer, UniHybri™ (Telechem International), ExpressHyb™Hybridization Solution (Clontech) and SlideHyb™ Survey Kit (Ambion), theaforementioned aqueous media mixed with organic solvents such as DMSOand DMF, the aforementioned aqueous media mixed with surfactants such asSDS (sodium dodecylsulfate), the aforementioned aqueous media mixed withvarious reagents including onium salts such as tetramethylammonium salt,formamide etc., which can change Tm of a nucleic acid to be hybridizedand so forth.

Further, for example, the device of the present invention can besuitably used in detection of SNP using LUMINEX System (Hitachi SoftwareEngineering Co., Ltd.), RT-PCR using GeneAmp 2400 (Perkin Elmer) orTP3000 (TAKARA), isolation of specimen using Te-MagS MBS (Tecan), mRNAIsolation Kit (Roche Diagnostics Corporation) or automatic plasmidextraction apparatus (TAKARA) and so forth.

In another embodiment, the device of the present invention is used fortreatment of diseases. In this embodiment, a biologically activesubstance functions as an active ingredient of a therapeutic agent.

EXAMPLES

The present invention will be explained more specifically with referenceto the following examples. However, the scope of the present inventionis not limited to these examples. In the following examples, “part”means “weight part”, and “water” means “distilled water” unlessotherwise indicated.

Example 1 Production of Core Particles

<1> Production Example 1 of Core Particle

A mixture comprising the following components was charged into a 500-mlflask in a batch, and dissolved oxygen was replaced with nitrogen. Then,the mixture was heated at 68° C. for about 15 hours on an oil bath withstirring using a stirrer under a nitrogen flow to obtain astyrene/methacrylic acid copolymer particle solution. Styrene  48.2parts Methacrylic acid  20.6 parts Methanol 179.8 parts Ethanol  29.9parts Water  59.8 parts Azobis-2-methylbutyronitrile (ABNE)  3.0 partsStyrene/methacrylic copolymer resin solution  75.0 parts(Styrene:2-hydroxyethyl Methacrylate=2:8, 40% by Weight Solution inMethanol)

Subsequently, a part of this particle solution was repeatedly washedwith a mixture of water and methanol (3:7) and filtered 3 to 5 times orso by using suction filtration equipment, and then vacuum-dried toobtain Core Particles 1. When the shapes of the particles were examinedby using SEM (S-2150, Hitachi, Ltd.), spherical particles were observed.The particle diameter was measured, and the average particle diameterwas found to be 1.42 μm.

<2> Production Example 2 of Core Particle

A mixture comprising the following components was charged into a 500-mlflask in a batch, and dissolved oxygen was replaced with nitrogen. Then,the mixture was heated at 70° C. for about 15 hours on an oil bath withstirring by using a stirrer under a nitrogen flow to obtainstyrene/methacrylic acid copolymer particle solution. Styrene  34.4parts Methacrylic acid  8.6 parts Methanol 208.0 parts Water  52.0 partsAzobis-2-methylbutyronitrile (ABNE)  1.0 part Polyvinylpyrrolidone(K-90)  15.0 parts

Subsequently, a part of this particle solution was repeatedly washedwith a mixture of water and methanol (3:7) and filtered 3 to 5 times orso by using suction filtration equipment, and then vacuum-dried toobtain Core Particles 2. When the shapes of the particles were examinedby using SEM (S-2150, Hitachi, Ltd.), spherical particles were observed.The particle diameter was measured, and the average particle diameterwas found to be 0.78 μm.

The results of the above production of core particles are summarized inTable 1. TABLE 1 Equivalent of Reactive group functional group containedin in polymer Raw material particles particles used Core Particle 1Carboxyl group 287/COOH Styrene, methacrylic acid Core Particle 2Carboxyl group 430/COOH Styrene, methacrylic acid

Example 2 Synthesis of Organic compound A

<1> Synthesis Example 1 of Polycarbodiimide Compound

In an amount of 500 g of 2,6-tolylene diisocyanate (TDI) and 367.8 g ofpolyoxyethylene monomethyl ether having a polymerization degree m of 8were initially reacted at 50° C. for 1 hour, then added with 5 g of acarbodiimidation catalyst and reacted at 85° C. for 6 hours to obtain apolycarbodiimide compound (polymerization degree=5) having blocked ends.This compound was gradually added with 508.3 g of distilled water toobtain a solution of Polycarbodiimide Compound 1 (resin concentration:60% by weight). The carbodiimide equivalent was 318/NCN.

<2> Synthesis Example 2 of Polycarbodiimide Compound

In an amount of 500 g of tetramethylxylylene diisocyanate (TMXDI) and 10g of a carbodiimidation catalyst were reacted at 180° C. for 10 hours toobtain poly-m-tetramethylxylylenecarbodiimide (polymerization degree=3)having an isocyanate end. This compound was added with 393.5 g ofpolyoxyethylene monomethyl ether having a polymerization degree m of 8and reacted at 140° C. for 6 hours to obtain Polycarbodiimide Compound 2having blocked ends. This compound was gradually added with 550.6 g ofdistilled water to obtain a solution of Polycarbodiimide Compound 2(resin concentration: 60% by weight). The carbodiimide equivalent was537/NCN.

<3> Synthesis Example 3 of Polycarbodiimide Compound

In an amount of 500 g of 2,6-tolylene diisocyanate (TDI) and 44.1 g ofethanol were initially reacted at 40° C. for 1 hour, then added with 5 gof a carbodiimidation catalyst and reacted at 75° C. for 7 hours toobtain a polycarbodiimide resin (polymerization degree=5) having blockedends. This compound was gradually added with 292.4 g of tetrahydrofuranto obtain a solution of Polycarbodiimide Compound 3 (resinconcentration: 60% by weight). The carbodiimide equivalent was 183/NCN.When a part of the obtained polycarbodiimide compound (polymerizationdegree=5) was examined for water-solubility, this polycarbodiimidecompound was hardly dissolved and found to be hydrophobic.

The results of the syntheses of polycarbodiimide compounds obtainedabove are summarized in Table 2. TABLE 2 Organic Functional Raw materialcompound, groups for blocked Functional Synthesis Average no. end groupExample per molecule segment equivalent Medium Synthesis CarbodiimidePolyoxyethylene 318 Water Example 1 group monomethyl 5 ether SynthesisCarbodiimide Polyoxyethylene 537 Water Example 2 group monomethyl 3ether Synthesis Carbodiimide Ethanol 183 THF Example 3 group 5

Example 3 Production of Base Particle (A)

<1> Production Example 1 of Base Particle

A mixture comprising the following components was charged into a 300-mlflask in a batch, heated at 45° C. for about 15 hours on an oil bathwith stirring using a stirrer under a nitrogen flow, and apolycarbodiimide compound was thereby reacted to obtain a base particlesolution. Solution of Core Particle 1  18.0 parts Solution ofPolycarbodiimide Compound 1  16.6 parts Water  31.2 parts Methanol 151.4parts

Subsequently, the base particle was repeatedly washed with a mixture ofwater and methanol (3:7) 3 times and methanol twice or so and filteredby using suction filtration equipment, and then vacuum-dried to obtainBase Particle 1. The shapes of the particles were examined by using SEM(S-2150, Hitachi, Ltd.), and the average particle diameter was found tobe 1.76 μm. Further, when the particles were examined by using a Fouriertransform infrared spectrophotometer (FT-IR8200PC, ShimadzuCorporation), the absorbance peak of the carbodiimide group was observedat a wavelength of about 2150 (1/cm).

Further, when a part of the particles were dispersed in water as amedium to confirm dispersibility on the basis of particle sizedistribution (Microtrac 9320HRA, NIKKISO Co., Ltd.), the particlediameter was found to be the same as the result obtained by SEM, whichwas a particle size suggesting monodispersion.

<2> Production Example 2 of Base Particle

A mixture comprising the following components was charged into a 300-mlflask in a batch, heated at 50° C. for about 15 hours on an oil bathwith stirring by using a stirrer under a nitrogen flow, and apolycarbodiimide compound was thereby reacted to obtain a base particlesolution. Solution of Core Particle 1 11.1 parts Solution ofPolycarbodiimide Compound 2  9.4 parts Water 28.2 parts Methanol 74.6parts

Subsequently, the base particle was repeatedly washed and filtered witha mixture of water and methanol (3:7) 3 times and methanol twice or sousing suction filtration equipment and then vacuum-dried to obtain BaseParticle 2. The shapes of the particles were examined by using SEM(S-2150, Hitachi, Ltd.), and the average particle diameter was found tobe 1.88 μm. Further, when this particle was examined by using a Fouriertransform infrared spectrophotometer (FT-IR8200PC, ShimadzuCorporation), the absorbance peak of the carbodiimide group was observedat a wavelength of about 2150 (1/cm).

Further, when a part of the particles were dispersed in water as amedium to confirm dispersibility on the basis of particle sizedistribution (Microtrac 9320HRA, NIKKISO Co., Ltd.), the particlediameter was found to be the same as the result obtained by SEM, whichwas a particle size suggesting monodispersion.

<3> Production Example 3 of Base Particle

A mixture comprising the following components was charged into a 300-mlflask in a batch, heated at 45° C. for about 15 hours on an oil bathwith stirring by using a stirrer under a nitrogen flow, and apolycarbodiimide compound was thereby reacted to obtain a base particlesolution. Solution of Core Particle 2  22.0 parts Solution ofPolycarbodiimide Compound 1  11.1 parts Water  20.9 parts Methanol 101.2parts

Subsequently, the base particle was repeatedly washed and filtered witha mixture of water and methanol (3:7) 3 times and methanol twice or sousing suction filtration equipment and then vacuum-dried to obtain BaseParticle 3. The shapes of the particles were examined by using SEM(S-2150, Hitachi, Ltd.), and the average particle diameter was found tobe 0.98 μm. Further, when the particles were examined by using a Fouriertransform infrared spectrophotometer (FT-IR8200PC, ShimadzuCorporation), the absorbance peak of the carbodiimide group was observedat a wavelength of about 2150 (1/cm).

Further, when a part of the particles were dispersed in water as amedium to confirm dispersibility on the basis of particle sizedistribution (Microtrac 9320HRA, NIKKISO Co., Ltd.), the particlediameter was found to be the same as the result obtained by SEM, whichwas a particle size suggesting monodispersion.

<4> Production Example 4 of Base Particle

A mixture comprising the following components was charged into a 300-mlflask in a batch, heated at 50° C. for about 15 hours on an oil bathwith stirring using a stirrer under a nitrogen flow, and apolycarbodiimide compound was thereby reacted to obtain a base particlesolution. Solution of Core Particle 2 22.1 parts Solution ofPolycarbodiimide Compound 2 12.5 parts Water 37.8 parts Methanol 99.8parts

Subsequently, the base particle was repeatedly washed and filtered witha mixture of water and methanol (3:7) 3 times and methanol twice or sousing suction filtration equipment and then vacuum-dried to obtain BaseParticle 4. The shapes of the particles were examined by using SEM(S-2150, Hitachi, Ltd.), and the average particle diameter was found tobe 1.06 μm. Further, when the particles were examined by using a Fouriertransform infrared spectrophotometer (FT-IR8200PC, ShimadzuCorporation), an absorbance peak of the carbodiimide group was observedat a wavelength of about 2150 (1/cm).

Further, when a part of the particles were dispersed in water as amedium to confirm dispersibility on the basis of particle sizedistribution (Microtrac 9320HRA, NIKKISO Co., Ltd.), the particlediameter was found to be the same as the result obtained by SEM, whichwas a particle size suggesting monodispersion.

<5> Production Example 5 of Base Particle

In accordance with the method described in Japanese Patent Laid-open No.8-23975, Example 6, a mixture comprising the following components wascharged into a 300-ml flask in a batch, and immersion was performed forabout 30 minutes with stirring using a stirrer. Cross-linked particles 5.0 parts (main component: divinyl benzene)* Solution ofPolycarbodiimide Compound 3 16.7 parts THF 83.3 parts*Cross-linked particles (SP-2095 produced by Sekisui fine Chemical Co.Ltd., average particle diameter: 9.5 μm, CV value: 4.7%)

Subsequently, the aforementioned mixture was filtered by using suctionfiltration equipment and dried by using a drier at temperature of 60° C.for about 3 hours to obtain polycarbodiimide compound-coated particles.

When the particles were examined by using a Fourier transform infraredspectrophotometer (FT-IR8200PC, Shimadzu Corporation), an absorbancepeak of the carbodiimide group was observed at a wavelength of about2150 (1/cm).

However, when a part of the particles were dispersed in water as amedium to confirm dispersibility on the basis of particle sizedistribution (Microtrac 9320HRA, NIKKISO Co., Ltd.), a distributionrepresented by one peak curve having a larger width and longerdistribution tails compared with particle diameter distribution of thebase particles of Production Examples 1 to 4 was obtained. When theshapes of the particles were examined by using SEM (S-2150, Hitachi,Ltd.), the average particle diameter was found to be 17.06 μm, and someaggregated particles were observed. When the average particle diameterwas calculated, aggregated particles were assumed as one particle, andan average value of the largest particle diameter and the smallestdiameter obtained with SEM was calculated as a particle diameter.

The results of the above production of base particles are summarized inTable 3. TABLE 3 Addition amount of Organic Base compound A ParticleCore (equivalent ratio Synthesis Dispersibility Production particle asto functional temperature in water Example used group) (° C.) as medium1 Production 3 45 ∘ Example 1 2 Production 2 50 ∘ Example 1 3 Production3 45 ∘ Example 2 4 Production 2 50 ∘ Example 2 5 — — — Δ∘: Monodispersed base particlesΔ: Partially monodispersed aggregated base particlesx: Base particles mostly consisting of aggregated particles

Example 4 Evaluation of Core Particles and Base Particles

<1> Evaluation Test 1

Base particles 1 to 5 produced in Example 3 and the core particles usedin production of these particles were each photographed by using SEMwith a magnification enabling measurement (×100 to 10,000), and themajor axis and the minor axis were randomly measured 15 times for oneparticle. This measurement was repeatedly performed for randomlyselected particles (n=100). Then, the average values were calculated forthe measured results, and a spherical particle exponential mean (majoraxis/minor axis) was calculated. The results are shown in Table 4. TABLE4 Base particle Spherical particle Spherical particle Productionexponential mean exponential mean Monodispersion, Example for coreparticles for base particles sphericalness 1 1.04 1.03 ∘ 2 1.04 1.05 ∘ 31.05 1.05 ∘ 4 1.05 1.06 ∘∘: Highly precise particles of which precipitation rate, particlesurface area and addition amount of biologically active substance can beequalizedx: Poor precision particles of which precipitation rate, particlesurface area and addition amount of biologically active substance aredifficult to be equalized<2> Evaluation Test 2

The base particles produced in Example 3 and the core particles used forproduction of these particles were each photographed by using SEM (×100to 10,000), the particle diameters were measured for randomly selectedparticles (n₁=500), and the average particle diameter was calculated.Then, the average thickness diameter (L) of the carbodiimide compoundlayer was calculated according to the following equation. The resultsare shown in Table 5.L=(L _(2−L) ₁)/2

L₁ is the average particle diameter of the experimentally produced coreparticles (A₁).

L₂ is the average particle diameter of the experimentally produced baseparticles (A). TABLE 5 Base Particle Average particle Average particleProduction diameter of core diameter of base Example particles (μm)particles (μm) L (μm) 1 1.42 1.76 0.17 2 1.42 1.88 0.23 3 0.78 0.98 0.104 0.78 1.06 0.14 5 9.5 17.06 ******: Numerical measurement was impossible due to aggregation ofparticles<3> Evaluation Test 3

CV values and CV ratios were calculated from the measurement results forthe base particles and the core particles obtained in the aforementionedevaluation. The results are shown in Table 6. TABLE 6 Base Particle CV₁value CV₂ value Production (%) of core (%) of base CV_(a) ratio Exampleparticle particle CV1/CV2 1 4.58 4.32 1.06 2 4.58 4.26 1.08 3 6.06 5.941.02 4 6.06 5.82 1.04 5 4.7  33.54  0.14

From the results of Evaluation Tests 1 to 3 described above, it wasconfirmed that the base particles having Organic compound A used in theexamples of the present invention were base particles having a layer ofOrganic compound A on the surface layer portions of the core particles,and were spherical particles having relatively even particle sizes.

Further, Base Particles obtained in the above production example 1 to 4were dispersed in water as a solvent by using known dispersion equipmentto form 1 weight % particle aqueous dispersions as solutions of BaseParticle 1 to 4. The solutions of Base Particles 1 to 4 were examined byusing a particle size distribution analyzer (Microtrac 9320HRA, NIKKISOCo., Ltd.), and it was found that the particles had average particlediameters comparable to those of the aforementioned particles, and asfor distribution, they were monodispersed particles of whichdistribution was represented by a sharp one-peak curve.

Example 5

<1> Production of Device

Oligonucleotides (30-mers) having the nucleotide sequences of SEQ IDNOS: 1, 2 and 3 were synthesized by using an oligonucleotide synthesizer(Perkin-Elmer Applied Biosystems) in a conventional manner. Theoligonucleotide of SEQ ID NO: 1 was biotinylated at the 5′ end. Theoligonucleotide of SEQ ID NO: 2 was complementary to a biotinylatedprobe (SEQ ID NO: 4), and the oligonucleotide of SEQ ID NO: 3 differedfrom the oligonucleotide of SEQ ID NO: 2 by one nucleotide and thus wasnot complementary thereto. These oligonucleotides were each dissolved in3×SSC at 100 pmol/μl.

Each of the aforementioned oligonucleotide solutions was mixed with thesolution of Base Particle 1 mentioned above in a quartz cell. Then, themixture was irradiated with an ultraviolet ray at 1200 mJ/cm² from 16 cmaway by using Uvstratalinker 2400 (STRATAGENE). The irradiation time was480 seconds. Then, the base particles were washed by shaking in waterfor 30 minutes and dried to obtain devices.

<2> Hybridization

The aforementioned devices and 60 μl of hybridization solution (ArrayitUniHyb (TeleCHem International, Inc.) containing 3 pmol of thebiotinylated probe (SEQ ID NO: 4, 262 bp) were mixed in an Eppendorftube and heated at 45° C. for 2 hours by using a drier. Theoligonucleotide of SEQ ID NO: 2 contained a sequence complementary tothe biotinylated probe (SEQ ID NO: 4).

Following the aforementioned hybridization, post-hybridization washingwas performed under the following conditions to remove the biotinylatedprobes non-specifically adsorbed on the devices.

[Conditions for Post-Hybridization Washing]

1) 2×SSC, 0.1% SDS, room temperature, 5 minutes, twice

2) 0.2×SSC, 0.1% SDS, 40° C., 5 minutes, twice

3) 2×SSC, room temperature, 1 minute, 3 times

In an amount of 1.5 ml of a blocking solution containing lactoproteins(Block Ace, Snow Brand Milk Products Co., Ltd.) was placed on thedevices to perform blocking at room temperature for 30 minutes. Theblocking solution was removed, and then 1.5 ml of astreptavidin/alkaline phosphatase conjugate solution (VECTOR) was placedand reacted at room temperature for 30 minutes. Subsequently, thedevices were immersed in a TBST solution (50 mM Tris-HCl (pH 7.5), 0.15M NaCl, 0.05% Tween 20) and shaken for 5 minutes to remove unreactedconjugates. Finally, the devices were added with 1.5 ml of a substratesolution (TMB) and left for 30 minutes to allow color developmentreaction.

The results are shown in Table 7. The signals of the particles on whichthe oligonucleotide of SEQ ID NO: 2 was immobilized represent the amountof the immobilized oligonucleotide. The signals of the particles onwhich the oligonucleotide of SEQ ID NO: 3 was immobilized representintensity of hybridization.

Comparative Example 1

<1> Production of Device

A solution of Base Particle (base particles produced according to themethod described in Japanese Patent Laid-open No. 8-23975, Example 6)produced in Example 3, Production Example 5 of Base Particle and each ofthe oligonucleotide solutions prepared in Example 5 were mixed in aquartz cell. Then, the mixture was irradiated with an ultraviolet ray at1200 mJ/cm² from 16 cm away by using Uvstratalinker 2400 (STRATAGENE).The irradiation time was 480 seconds. Then, the base particles werewashed in water for 30 minutes with shaking and then dried to obtaindevices.

<2> Hybridization

The aforementioned devices and 60 μl of hybridization solution (ArrayitUniHyb, TeleCHem International, Inc.) containing 3 pmol of biotinylatedprobe (SEQ ID NO: 4, 262 bp) were mixed in an Eppendorf tube and heatedat 45° C. for 2 hours by using a drier.

Following the aforementioned hybridization, hybridization was detectedin the same manner as in Example 5. The results are shown in Table 7.TABLE 7 Immobilized oligonucleotide SEQ ID NO: 1 SEQ ID NO: 2 SEQ ID NO:3 Example 5 ⊚ ⊚ x Comparative Δ Δ x Example 1⊚: Very clear signals were observed with very high sensitivity.∘: Clear signals were observed with high sensitivity.Δ: Signals were observed with low sensitivity or unclearly.x: No signal was observed.

As demonstrated by the results shown in Table 7, it was found that theoligonucleotides were reliably immobilized on the base particles in thedevices of Example 5. Further, in the devices of Example 5, thehybridization signals were also clearly observed. No signal was observedfrom the oligonucleotide of SEQ ID NO: 3. On the other hand, in thedevices of Comparative Example 1, the signals from the immobilizedoligonucleotides and hybridization signal were observed unclearly withlow sensitivity, and therefore it is considered that the carbodiimidecompound was fallen off from the base particles. Therefore, it isconsidered that, in the devices of Example 5, the carbodiimide compoundwas prevented from falling off from the base particles by the formationof covalent bonds between the base particles and the carbodiimidecompound, and as a result, a clear signal could be obtained with highsensitivity.

When devices were produced by immobilizing oligonucleotides on the baseparticles using the solution of Base Particles 2 to 4, and hybridizationwas detected in a similar manner, results similar to those of Example 5were obtained.

Example 6 Evaluation of Dispersibility of Devices

The devices obtained in Example 5 and Comparative Example 1 were dilutedwith water, and dispersibility was confirmed by using a particle sizedistribution analyzer (Microtrac 9320HRA, NIKKISO Co., Ltd.).

As a result, the devices of Example 5 were particles showingmonodispersion distribution similar to that of Base Particle 1, and nochange indicating a different distribution was observed. On the otherhand, the distribution of the devices immobilized with theoligonucleotide of Comparative Example 1 was wider than the particlediameter distribution of the used core particles, and it was representedby a one-peak curve with long distribution tails.

Further, as confirmed by using SEM (×100 to 10,000), aggregatedparticles and deformed particles (including the shapes of baseparticles) were not observed among the devices of Example 5, whereassome aggregated particles were observed among the devices of ComparativeExample 1. These results are shown in Table 8, and the device diameters,CV values and CV ratios are shown in Table 9. TABLE 8 Dispersibilitybased on particle size Aggregation/deformation distribution observed bySEM Device of Example 5 A No aggregated/deformed particles Device ofComparative B Aggregated particles Example 1A: Monodispersed devices having a particle diameter similar to that ofthe base particlesB: Devices a part of which had a particle diameter similar to that ofthe base particles, but which had a wide distribution rangeC: Devices not having a particle diameter similar to that of the baseparticles and having a wide distribution range.

TABLE 9 Device average Device particle CV₃ value CV_(b) ratio CV_(c)ratio diameter (μm) (%) CV₁/CV₃ CV₂/CV₃ Example 5  1.81  4.64 0.99 0.93Comparative 21.72 64.38 0.07 0.52 Example 1**Aggregated particles were assumed as one particle, and average of themajor axis and the minor axis of the particles was obtained by using SEMas the diameter of the particle.

This confirmed that the devices of the present invention had favorablesolution dispersibility and high performance.

When dispersibility was examined for the devices produced by using BaseParticles 2 to 4, it was confirmed that they showed favorable particlesize distribution and shape observed by using SEM and exhibitedfavorable dispersibility in a solution.

INDUSTRIAL APPLICABILITY

The device of the present invention shows good stability for dispersionin a solution and can be suitably used for detection or measurement of abiologically active substance or for therapeutic treatment.

1. A biologically active substance-immobilized device, which comprises abase particle comprising a core particle and an organic compound havingtwo or more hydrophilic groups and immobilized on the core particle by achemical bond and a biologically active substance bonded to the baseparticle via the organic compound.
 2. The device according to claim 1,monodispersed in an aqueous medium.
 3. The device according to claim 1,wherein the base particle has an average particle diameter of 0.01 to100 μm.
 4. The device according to claim 1, wherein the base particlehas a spherical or substantially spherical shape.
 5. The deviceaccording to claim 1, wherein at least one of CV_(b) ratio and CV_(c)ratio defined by the following equations is 0.6 to 3.0:CV _(b)ratio=CV ₁ /CV ₃CV _(c)ratio=CV ₂ /CV ₃CV ₁=(Standard deviation of core particle diameter/Average core particlediameter)×100CV ₂=(Standard deviation of base particle diameter/Average base particlediameter)×100CV ₃=(Standard deviation of device diameter/average device particlediameter)×100
 6. The device according to claim 1, wherein the coreparticle and the biologically active substance are bonded by a reactionwith a functional group selected from the group consisting ofcarbodiimide group, ester group, carbonate group, epoxy group andoxazoline group.
 7. The device according to claim 1, wherein the organiccompound is a compound represented by the following formula:A_(x)—(R—X)_(n)—R—A_(y)  (I) wherein A_(x) and A_(y) independentlyrepresent a segment having a functional group that exhibitshydrophilicity and may be identical or different, R independentlyrepresents an organic group of two or more valences, X independentlyrepresents carbodiimide group, epoxy group or oxazoline group, and n isan integer of 2 to
 80. 8. The device according to claim 1, wherein thebiologically active substance is selected from a nucleic acid, protein,hapten and saccharide.
 9. The device according to claim 1, which is fordetecting or measuring a second biologically active substance containedin a sample by using a specific bond of the biologically activesubstance and the second biologically active substance in the sample.10. The device according to claim 1, wherein the biologically activesubstance is an agent for therapeutic treatment of a disease.
 11. Thedevice according to claim 7, wherein n is an integer of 2 to
 40. 12. Amethod of detecting or measuring a second biologically active substancein a sample comprising the step of binding the second biologicallyactive substance to the biologically active substance bound to the baseparticle in the device of claim 1.